CN107384796B - Method for enriching and separating microorganisms from extreme acid environment sample, corresponding culture medium and preparation method - Google Patents
Method for enriching and separating microorganisms from extreme acid environment sample, corresponding culture medium and preparation method Download PDFInfo
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Abstract
The invention discloses a method for enriching and separating microorganisms from an extremely acidic environment sample, a corresponding culture medium and a preparation method. The invention discloses a microorganism liquid enrichment culture medium and a microorganism solid plate culture medium, which can be used for separating microorganisms from samples in an extremely acidic environment. The invention relates to enrichment, separation and identification of microbes in an extreme acid environment by simulating a multi-trace-element culture medium in the extreme acid environment, and the enrichment and separation method can increase the individual number of certain rare microbe groups, thereby obviously improving the separation probability of the rare microbe groups, increasing the microbial diversity of separation results, and having important value and significance for identification and research of microbes in the extreme acid environment.
Description
Technical Field
The invention belongs to the technical field of extreme environment microorganism separation, and particularly relates to a method for enriching and separating microorganisms in acid mine wastewater, a corresponding culture medium and a preparation method.
Background
Acid Mine Drainage (AMD), which generally refers to the leach water of waste ores and tailings, exhibits extremely low acidity (pH <3), contains high concentrations of metals (Pb, Zn, Cu, Mn, Cd, As, Fe, Al, etc.) and non-metallic ions, is considered to be an extremely acidic environment, and is also a serious pollution problem commonly faced by the global mining industry. The acidic wastewater can cause destructive damage to surrounding land and water ecosystems, so that most of animals, plants, fishes, algae and other organisms die, and the survival of human beings is directly harmed. The problem of acidic mine wastewater pollution in China is quite serious, about 10 percent of the cultivated land area in China is polluted by heavy metal (about 1.5 million acres), and the direct economic loss caused by the heavy metal polluted grain reaches 1200 million tons every year exceeds 200 million yuan. The Guangdong is the forefront of economic development, the problems of serious pollution and ecological damage of a mining area exist for more than 20 years, and a large amount of mine acidic wastewater containing toxic heavy metal ions is directly discharged into a downstream river without treatment, so that the ecological system of the mining area and the river along the bank is destructively damaged. Therefore, the treatment of AMD pollution is a global major environmental problem to be solved urgently and is also an urgent task faced by the current environmental treatment in China.
Compared with other treatment technologies (artificial wetland method and neutralization method), the microbial treatment technology for extremely acidic environmental pollution such as mine acidic wastewater has the advantages of low cost, no secondary pollution, capability of controlling soil or wastewater acidification from the source and the like, and iron reducing bacteria (FRB) and Sulfate Reducing Bacteria (SRB) are mostly adopted to treat acidification problems in the existing practical application case. Therefore, the separation of more microbial resources with such biological functions from such extreme acidic environments helps us to further accelerate the resolution of such environmental problems.
Due to the extreme acidity of the acidic mine wastewater environment, the microbial community structure is simple, and in the early stage of microbial community research of the acidic mine wastewater, the main groups of microorganisms in the environment comprise Proteobacteria (Proteobacteria), Nitrospira (Nitrospira), firmicutes (Fimicutes) and Acidobacteria (Acidobacteria). Relatively few studies are made on archaea in acid mine wastewater, and the archaea branches mainly relate to thermoplasiales and sulfolobules. In recent years, some studies have found that the diversity of eukaryotes is significantly higher in some extreme acidic environments than that of prokaryotes. For example, Spanish Rio Tinto River is a typical acidic mine wastewater environment with more eukaryotic diversity and biomass than prokaryotic organisms. The combined action of the microorganisms accelerates the oxidation of ores and causes huge environmental pollution, and the most effective method for treating acid mine wastewater is a biological prevention strategy at present. Therefore, the deep understanding and mastering of the microbial groups and the diversity thereof in the acidic mine wastewater environment are beneficial to the later-stage bioremediation by using related microorganisms, and have great practical production significance.
The traditional microbial research method in acidic environment, namely a pure culture technology, comprises selection, separation, culture, physiological identification and the like, and the method is simple but has great limitation. Because the living conditions of the environment are very special (pH <3), the species of microorganisms which can be cultured in pure form belong to a few. Firstly, most heterotrophic microorganisms need to undergo redox potential conversion by means of metal ions in the environment, pure culture under laboratory conditions, different from the original ecological environment in which the heterotrophic microorganisms grow in nutritional conditions, temperature, humidity and the like, and the types and the number of the obtained microorganisms are also changed. Secondly, fungi and algae (autotrophic microorganisms) grow slowly and are highly sensitive to organic matter and are difficult to obtain in pure culture processes. In addition, the conventional solid isolation medium cannot be isolated and cultured under extremely low pH conditions. At present, the knowledge of the microbial community structure of the extreme acid environment such as acid mine wastewater is still very limited, and the microbial community structure is mainly based on metagenome, macrotranscriptome and a small amount of pure culture microorganisms, however, the microbial community in the environment still has a large amount of pure culture which is not obtained, and the research and application of the microbial community structure are far behind that of other extreme environment microorganisms. Therefore, there is a need to solve the technical problem of pure culture, so as to obtain pure culture of microorganisms in the environment as much as possible, and to promote the study of microbial community composition of the environment, and even the remediation of acid mine wastewater environment.
Disclosure of Invention
The invention aims to: the method solves the problems of limited understanding of the structure of the microbial community of the extreme acid environment such as the acid mine wastewater and the like, difficult separation and the like, and provides a method for remarkably improving the species and the quantity of the microorganisms separated from the extreme acid environment on the existing pure culture level.
In order to achieve the above object, the present invention provides a microorganism liquid enrichment medium, which comprises the following components:
per liter of solution A: MgSO (MgSO)4·7H2O 0.70g，(NH4)2SO41.80g，K2S4O60.76g，TSB 0.25g， pH2.5；
Per liter of solution B: FeSO4·7H2O 7g，MnCl2-4H2O 1.8mg，Na2B4O7·10H2O 4.5mg， ZnSO4-7H2O 0.22mg，CuCl2-2H2O 0.05mg，NaMoO4-2H2O 0.03mg，VOSO4-2H2O 0.03mg，CoSO40.01mg，pH2.0。
The preparation method of the microorganism liquid enrichment culture medium comprises the following steps: sealing the solution A, sterilizing at 121 ℃ for 25min, adding the solution B after filtration and sterilization, and mixing to obtain a microorganism liquid enrichment culture medium; the volume ratio of the solution A to the solution B is 17: 1; the water used for preparing the solution A and the solution B is acid mine wastewater after filtration and sterilization.
As a preferable technical scheme of the preparation method of the microorganism liquid enrichment medium, the filtration sterilization is filtration by using a 0.22 mu m filter membrane.
In order to achieve the above object, the present invention also provides a microorganism solid plate culture medium comprising the following components:
per liter of solution A: MgSO (MgSO)4·7H2O 0.70g，(NH4)2SO41.80g，K2S4O60.76g，TSB 0.25g， pH2.5；
Per liter of solution B: FeSO4·7H2O 7g，MnCl2-4H2O 1.8mg，Na2B4O7·10H2O 4.5mg， ZnSO4-7H2O 0.22mg，CuCl2-2H2O 0.05mg，NaMoO4-2H2O 0.03mg，VOSO4-2H2O 0.03mg，CoSO40.01mg，pH2.0；
Per liter of solution C: 80g of agarose.
The preparation method of the microorganism solid plate culture medium comprises the following steps: sealing the solution A and the solution C respectively, sterilizing at 121 deg.C for 25min, cooling to 55 deg.C, adding filtered and sterilized solution B, and mixing; the volume ratio of the solution A to the solution B to the solution C is 73: 2: 25; the water used for preparing the solution A and the solution B is acid mine wastewater after filtration and sterilization; and the water used for preparing the solution C is double distilled water.
As a preferable technical scheme of the preparation method of the microorganism solid plate culture medium, the filtration sterilization is filtration by using a 0.22 μm filter membrane.
In order to achieve the above object, the present invention also provides a method for isolating microorganisms from a sample in an extremely acidic environment, comprising the steps of:
(1) enriching an extreme acid environment sample by using the microorganism liquid enrichment culture medium to obtain an enriched sample;
(2) diluting and coating the enriched sample in the step (1) in gradient by using the microbial solid plate culture medium, and then placing the sample in different incubators for continuous culture;
(3) and classifying and identifying the obtained microorganisms to obtain the types and the number of finally separated microorganisms.
As a preferred technical scheme of the method for separating the microorganisms from the extreme acid environment sample, in the step (1), the enrichment is to add the extreme acid environment sample into the microorganism liquid enrichment culture medium, respectively place the sample in shaking tables at 28 ℃ and 37 ℃, and culture the sample in a dark place at 180rpm for 1 week; in the step (2), the continuous culture is carried out at the temperature of 28 ℃ and the temperature of 37 ℃ which are adopted when corresponding enrichment is carried out, and the plate is placed in an incubator in an inverted mode and is not protected from light for 14-28 days.
As a preferred embodiment of the method for separating microorganisms from a sample in an extremely acidic environment, step (2), the step of diluting with gradient and coating is carried out at 10 degrees-1、10-2Two dilution gradients, coated separately.
As a preferable technical scheme of the method for separating the microorganisms from the extreme acid environment sample, the extreme acid environment sample is acid mine wastewater.
Classifying and identifying the obtained microorganisms, and selecting three universal primers of 18S, ITS and 16S rDNA for amplification and sequencing, wherein for gene extraction of eukaryote, a liquid nitrogen grinding method can be adopted.
Compared with the prior art, the invention has the following beneficial effects:
the invention relates to enrichment, separation and identification of microbes in an extreme acid environment by simulating a multi-trace-element culture medium in the extreme acid environment, and the enrichment and separation method can increase the individual number of certain rare microbe groups, thereby obviously improving the separation probability of the rare microbe groups, increasing the microbial diversity of separation results, and having important value and significance for identification and research of microbes in the extreme acid environment.
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The invention and the advantages thereof are explained in detail with reference to the accompanying drawings and the detailed description.
FIG. 1 shows the results of the enrichment of a sample by the method of the present invention.
FIGS. 2 to 3 show the results of plate separation and the repetition thereof in the method of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantageous technical effects of the present invention clearer, the present invention is further described in detail with reference to the following embodiments. It should be understood that the examples described in this specification are for the purpose of illustration only and are not intended to limit the invention, and the formulation, proportions, etc. of the examples may be selected appropriately without materially affecting the results.
Example 1
Preparing a microorganism liquid enrichment medium:
per liter of solution A: MgSO (MgSO)4·7H2O 0.70g，(NH4)2SO41.80g，K2S4O60.76g, TSB 0.25g, pH2.5; placing 17ml LA solution in a conical flask, sealing, and sterilizing at 121 ℃ for 25 minutes;
per liter of solution B: FeSO4·7H2O 7g，MnCl2-4H2O 1.8mg，Na2B4O7·10H2O 4.5mg， ZnSO4-7H2O 0.22mg，CuCl2-2H2O 0.05mg，NaMoO4-2H2O 0.03mg，VOSO4-2H2O 0.03mg，CoSO40.01mg, pH2.0; the 1mLB solution was filtered through a 0.22 μm filter membrane and sterilized, and added to the sterilized solution A, and the set was repeated 2 times.
The used water is obtained by filtering the acidic mine wastewater of Minkou lead-zinc ore in Mingxiao county of Guangdong province, China with a 0.22-micron filter membrane.
Preparing a microorganism solid plate culture medium:
per liter of solution A: MgSO (MgSO)4·7H2O 0.70g，(NH4)2SO41.80g，K2S4O60.76g, TSB 0.25g, pH2.5; placing 730mLA solution in a conical flask, sealing, and sterilizing at 121 ℃ for 25 minutes;
per liter of solution B: FeSO4·7H2O 7g，MnCl2-4H2O 1.8mg，Na2B4O7·10H2O 4.5mg， ZnSO4-7H2O 0.22mg，CuCl2-2H2O 0.05mg，NaMoO4-2H2O 0.03mg，VOSO4-2H2O 0.03mg，CoSO40.01mg, pH2.0; filtering 20mL of the solution B with a 0.22-micron filter membrane for sterilization;
per liter of solution C: agarose 80g, 1L double distilled water; placing 250mL of the solution C in a conical flask, sealing, and sterilizing at 121 ℃ for 25 minutes.
Mixing the sterilized solution A, solution C and the sterilized solution B, and pouring the mixture into a flat plate.
Example 2
The method comprises the steps of taking acid mine wastewater of Van kou lead-zinc ore in Mingxiao city, Guangdong province, China as a sample (pH is less than 3), carrying out microbial enrichment on microbial resources and an ecological laboratory of life sciences academy of Zhongshan university by using a microbial liquid enrichment medium, coating the sample after 1 week, inversely placing the sample in an incubator for isolated culture, obtaining a single colony after 14-28 days, and carrying out 18S, ITS and 16S rDNA sequencing identification to count the pure culture type and quantity of microorganisms in the Van kou sample. In the completed ITS, 18S rRNA and 16S rRNA gene identification results, 26 bacteria, 6 fungi and 1 alga have been obtained, and the data is still in continuous renewal. The number of pure culture microorganisms of the sample obtained by the method is far greater than that of other published culture media and methods thereof, and is also far greater than that of a direct coating plate culture method without enrichment.
TABLE 1 microorganisms isolated from acidic mine wastewater of Guangdong Minkou lead-zinc ore
The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications that do not depart from the scope of the present invention are intended to be included within the scope of the present invention.
Claims (9)
1. A microorganism liquid enrichment culture medium is characterized by comprising the following components:
per liter of solution A: MgSO (MgSO)4·7H2O 0.70g，(NH4)2SO4 1.80g，K2S4O6 0.76g，TSB 0.25g，pH2.5；
Per liter of solution B: FeSO4·7H2O 7g，MnCl2-4H2O 1.8mg，Na2B4O7·10H2O 4.5mg，ZnSO4-7H2O 0.22mg，CuCl2-2H2O 0.05mg，NaMoO4-2H2O 0.03mg，VOSO4-2H2O 0.03mg，CoSO4 0.01mg，pH2.0；
The water used for preparing the solution A and the solution B is the acid mine wastewater of lead-zinc ore after filtration and sterilization.
2. The method for preparing a liquid-enriched culture medium for microorganisms according to claim 1, comprising the steps of: sealing the solution A, sterilizing at 121 ℃ for 25min, adding the solution B after filtration and sterilization, and mixing to obtain a microorganism liquid enrichment culture medium; the volume ratio of the solution A to the solution B is 17: 1; the water used for preparing the solution A and the solution B is acid mine wastewater of lead-zinc ore after filtration and sterilization.
3. The method for preparing a culture medium enriched in a liquid of a microorganism as claimed in claim 2, wherein the filtration sterilization is a filtration using a 0.22 μm filter.
4. A microorganism solid plate culture medium is characterized by comprising the following components:
per liter of solution A: MgSO (MgSO)4·7H2O 0.70g，(NH4)2SO4 1.80g，K2S4O6 0.76g，TSB 0.25g，pH2.5；
Per liter of solution B: FeSO4·7H2O 7g，MnCl2-4H2O 1.8mg，Na2B4O7·10H2O 4.5mg，ZnSO4-7H2O 0.22mg，CuCl2-2H2O 0.05mg，NaMoO4-2H2O 0.03mg，VOSO4-2H2O 0.03mg，CoSO4 0.01mg，pH2.0；
Per liter of solution C: 80g of agarose.
The water used for preparing the solution A and the solution B is the acid mine wastewater of lead-zinc ore after filtration and sterilization.
5. The method for preparing a microbial solid plate culture medium according to claim 4, comprising the steps of: sealing the solution A and the solution C respectively, sterilizing at 121 deg.C for 25min, cooling to 55 deg.C, adding filtered and sterilized solution B, and mixing; the volume ratio of the solution A to the solution B to the solution C is 73: 2: 25; the water used for preparing the solution A and the solution B is acid mine wastewater of lead-zinc ore after filtration and sterilization; and the water used for preparing the solution C is double distilled water.
6. The method for preparing a solid plate culture medium of microorganisms according to claim 5, wherein the filtration sterilization is filtration using a 0.22 μm filter.
7. A method for isolating microorganisms from a sample in an extremely acidic environment, comprising the steps of:
(1) enriching a sample in an extremely acidic environment by using the microbial liquid enrichment medium of claim 1 to obtain an enriched sample;
(2) diluting and coating the enriched sample in the step (1) by gradient by using the microbial solid plate culture medium of claim 4, and then placing the sample in different incubators for continuous culture;
(3) and classifying and identifying the obtained microorganisms to obtain the types and the number of finally separated microorganisms.
The extreme acid environment sample is acid mine wastewater of lead-zinc ores.
8. The method for separating microorganisms from extreme acidic environment samples according to claim 7, wherein in the step (1), the enrichment is carried out by adding the extreme acidic environment samples into the microorganism liquid enrichment medium, respectively placing the sample in shaking tables at 28 ℃ and 37 ℃, and culturing the sample for 1 week at 180rpm without light shielding; in the step (2), the continuous culture is carried out at the temperature of 28 ℃ and the temperature of 37 ℃ which are adopted when corresponding enrichment is carried out, and the plate is placed in an incubator in an inverted mode and is not protected from light for 14-28 days.
9. The method for separating microorganisms from samples in extremely acidic environments as claimed in claim 7, wherein in the step (2), the step of diluting and coating with gradient is 10-1、10-2Two dilution gradients, coated separately.
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| CN101792728A (en) * | 2010-04-01 | 2010-08-04 | 中国科学院微生物研究所 | Method for leaching metal from metallic ores and special strain in same |
| CN102876702A (en) * | 2012-10-15 | 2013-01-16 | 中国科学院微生物研究所 | Shuttle expression vector with broad hosts |
| CN103966127A (en) * | 2014-04-28 | 2014-08-06 | 河南师范大学 | Rhodococcuscercidiphylli and screening method and application thereof |
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| CN101792728A (en) * | 2010-04-01 | 2010-08-04 | 中国科学院微生物研究所 | Method for leaching metal from metallic ores and special strain in same |
| CN102876702A (en) * | 2012-10-15 | 2013-01-16 | 中国科学院微生物研究所 | Shuttle expression vector with broad hosts |
| CN103966127A (en) * | 2014-04-28 | 2014-08-06 | 河南师范大学 | Rhodococcuscercidiphylli and screening method and application thereof |
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